Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering

Jihyeon Yu; Eunju Cho; Yeon-Gil Choi; You Kyeong Jeong; Yongwoo Na; Jong-Seo Kim; Sung-Rae Cho; Jae-Sung Woo; Sangsu Bae

doi:10.1021/acssynbio.0c00090

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering

ACS Synth Biol. 2020 Jul 17;9(7):1591-1598. doi: 10.1021/acssynbio.0c00090. Epub 2020 Jul 2.

Authors

Jihyeon Yu^{1

2}, Eunju Cho^{3

4}, Yeon-Gil Choi⁵, You Kyeong Jeong^{1

2}, Yongwoo Na^{6

7}, Jong-Seo Kim^{6

7}, Sung-Rae Cho^{3

4

8}, Jae-Sung Woo⁵, Sangsu Bae^{1

2}

Affiliations

¹ Department of Chemistry, Hanyang University, Seoul 04763, South Korea.
² Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea.
³ Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul 03722, South Korea.
⁴ Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, South Korea.
⁵ Department of Life Sciences, Korea University, Seoul 02841, South Korea.
⁶ Center for RNA Research, Institute for Basic Science (IBS), Seoul 08826, South Korea.
⁷ School of Biological Sciences, Seoul National University, Seoul 08826, South Korea.
⁸ Graduate Program of Nano Science and Technology, Yonsei University College of Medicine, Seoul 03722, South Korea.

PMID: 32584551
DOI: 10.1021/acssynbio.0c00090

Abstract

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

Keywords: CRISPR-Cas system; Reelin; genome engineering; molecular cloning; protein purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
Cell Adhesion Molecules, Neuronal / analysis
Cell Adhesion Molecules, Neuronal / genetics
Cell Adhesion Molecules, Neuronal / metabolism*
Cell Line, Tumor
Cell Survival / drug effects
Chromatography, High Pressure Liquid
Extracellular Matrix Proteins / analysis
Extracellular Matrix Proteins / genetics
Extracellular Matrix Proteins / metabolism*
Gene Editing / methods*
Humans
Microscopy, Fluorescence
Nerve Tissue Proteins / analysis
Nerve Tissue Proteins / genetics
Nerve Tissue Proteins / metabolism*
Plasmids / genetics
Plasmids / metabolism
RNA, Guide, CRISPR-Cas Systems / metabolism
Recombinant Proteins / analysis
Recombinant Proteins / biosynthesis
Recombinant Proteins / isolation & purification
Recombinant Proteins / pharmacology
Reelin Protein
Serine Endopeptidases / analysis
Serine Endopeptidases / genetics
Serine Endopeptidases / metabolism*
Tandem Mass Spectrometry

Substances

Cell Adhesion Molecules, Neuronal
Extracellular Matrix Proteins
Nerve Tissue Proteins
RNA, Guide, CRISPR-Cas Systems
Recombinant Proteins
Reelin Protein
RELN protein, human
Serine Endopeptidases